Anti-mesothelin immunotoxin SS1P in combination with gemcitabine results in increased activity against mesothelin-expressing tumor xenografts.

PURPOSE: To determine the antitumor activity of the anti-mesothelin immunotoxin SS1P in combination with gemcitabine against mesothelin-expressing tumor xenografts. EXPERIMENTAL DESIGN: The in vitro activity of SS1P in combination with gemcitabine against the mesothelin-expressing cell line A431/K5 was evaluated using cytotoxicity and apoptosis assays. The antitumor activity of this combination was evaluated in nude mice bearing A431/K5 tumor xenografts. Tumor-bearing mice were treated with different doses and schedules of gemcitabine alone, SS1P alone (0.2 mg/kg i.v. every other day x three doses), or with both agents together, and tumor volumes were measured over time. RESULTS: In vitro studies failed to show the synergy of SS1P plus gemcitabine against the mesothelin-expressing A431/K5 cells. In contrast, in the in vivo setting, there was a marked synergy when SS1P was combined with gemcitabine for the treatment of mesothelin-expressing tumor xenografts. This synergy was present using different doses and schedules of gemcitabine administration. In mice treated with fractionated doses of gemcitabine in combination with SS1P, complete tumor regression was observed in all mice and was long-lasting in 60% of the animals. Also, this antitumor activity was specific to SS1P because HA22, an immunotoxin targeting CD22 not expressed on A431/K5 cells, did not increase the efficacy of gemcitabine. CONCLUSIONS: SS1P in combination with gemcitabine results in marked antitumor activity against mesothelin-expressing tumors. This combination could be potentially useful for the treatment of human cancers that express mesothelin and are responsive to gemcitabine therapy.     

Tumor-directed radiation and the immunotoxin SS1P in the treatment of mesothelin-expressing tumor xenografts.

PURPOSE: Mesothelin is a cell surface protein overexpressed in mesotheliomas and pancreatic and ovarian cancers. The goal of this study was to determine if radiation therapy in combination with the antimesothelin immunotoxin SS1(dsFv)PE38 (SS1P) would result in enhanced antitumor activity against mesothelin-expressing xenografts in nude mice. EXPERIMENTAL DESIGN: Female athymic nude mice bearing s.c. mesothelin-expressing xenografts were treated with SS1P alone, tumor-focused radiation alone, or the combination of the two. Two different regimens of the combination therapy were tested. In the low-dose combination schedule, mice were treated with either 5 Gy radiation alone, 0.2 mg/kg SS1P alone, or the same doses of radiation and SS1P in combination. In the high-dose combination experiments, mice were treated with either 15 Gy radiation alone, 0.3 mg/kg SS1P alone, or the combination of radiation and SS1P. RESULTS: In the low-dose radiation and SS1P combination studies, mice treated with the combination of radiation and SS1P had a statistically significant prolongation in time to tumor doubling or tripling compared with control, SS1P, or radiation alone. A similar increase in time to tumor doubling or tripling was seen in mice treated with high-dose radiation and SS1P combination. CONCLUSIONS: Combination of SS1P with tumor-directed radiation results in enhanced antitumor activity against mesothelin-expressing tumor xenografts. This effect was seen when either low or high doses of radiation were used.     

Complete regression of human B-cell lymphoma xenografts in mice treated with recombinant anti-CD22 immunotoxin RFB4(dsFv)-PE38 at doses tolerated by cynomolgus monkeys

RFB4(dsFv)-PE38 is a recombinant immunotoxin in which the variable light domain (V(L)) is disulfide bonded via cysteine residues to the variable heavy domain (V(H)), which in turn is fused to PE38, a mutant form of Pseudomonas exotoxin A. RFB4 binds to CD22, which is a differentiation antigen expressed on the majority of B-cell leukemias and lymphomas. To examine the potential efficacy of RFB4(dsFv)-PE38 when administered at a dose schedule appropriate for phase I testing, mice bearing CA46 human CD22+ Burkitt's lymphoma xenografts were treated on alternate days i.v. for 3 doses (QOD x 3). Complete regressions were observed in 80% and 100% of mice treated with 200 and 275 microg/kg QOD x 3, respectively. The higher dose was 27% of the LD50 and 34% of the LD10 in mice. Because RFB4(dsFv)-PE38 is stable at 37 degrees C, it could also be given by continuous infusion using pumps placed in the peritoneal cavity; complete regressions also resulted from this mode of administration. To study toxicology, a pilot toxicology study of RFB4(dsFv)-PE38 was undertaken in cynomolgus monkeys, which like humans but unlike mice have CD22, which binds RFB4. Doses of 100 and 500 microg/kg i.v. QOD x 3 were well tolerated, indicating that a dose that cured tumors in mice was tolerated by primates. Based on these preclinical results, RFB4(dsFv)-PE38 is being developed for the treatment of patients with CD22-positive leukemias and lymphomas.     

HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity.

PURPOSE: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue-resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22. EXPERIMENTAL DESIGN: Arg(490), which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured. RESULTS: HA22 (R490A) is approximately 2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD(50)s (approximately 1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity. CONCLUSION: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.     

Cytotoxic activity of disulfide-stabilized recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignant cells from patients with B-cell leukemias.

Chemical conjugates of anti-CD22 monoclonal antibodies and toxins have been used to treat CD22+ hematological malignancies. A new anti-CD22 recombinant immunotoxin RFB4(dsFv)-PE38, composed of the Fv portion of the monoclonal antibody RFB4 fused to a truncated form of Pseudomonas exotoxin A, is being developed to target CD22+ tumor cells. To explore the potential clinical utility of this recombinant toxin in treating patients with B-cell malignancies, the fresh cells of patients were incubated ex vivo with RFB4(dsFv)-PE38. Specific cytotoxicity was demonstrated in the malignant cells of 25 of 28 patients with a variety of B-cell malignancies, including acute and chronic lymphocytic leukemias and large cell, mantle cell, and follicular lymphomas. The IC50S, the concentrations necessary for 50% inhibition of protein synthesis, were 3-10 ng/ml in five patients and 10-50 ng/ml in seven patients. Cytotoxicity correlated with cell death upon direct examination of the malignant cells. Significant cytotoxicity was observed with cells containing as few as 350 CD22 sites/cell. A more active derivative of RFB4(dsFv)-PE38, RFB4(dsFv)-PE38KDEL, was produced and was slightly to more than 10-fold more cytotoxic toward patient cells and about twice as toxic to mice. Thus, RFB4(dsFv)-PE38 was specifically cytotoxic toward malignant cells from patients with B-cell leukemias. These data support the testing of RFB4(dsFv)-PE38 in patients with CD22+ leukemias and lymphomas, which is presently under way.     

Administration of disulfide-stabilized Fv-immunotoxins B1(dsFv)-PE38 and B3(dsFv)-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice

B1 (dsFv)-PE38 and B3(dsFv)-PE38 are recombinant immunotoxins in which the Fv fragments of MAbs B1 and B3, respectively, are stabilized by an engineered interchain disulfide bond and are fused at their C-termini to a modified Pseudomonas exotoxin from which the cell-binding domain has been deleted (PE38). Both immunotoxins have been shown to be specifically cytotoxic toward human cancer cell lines which express Le^Y-related carbohydrates on their surface, and when given i.v., eradicated 30-to 50-mm³ s.c. A431 tumors growing in nude mice. A major advantage of dsFv-immunotoxins is their stability at 37°C compared with the relatively unstable single-chain Fvs. This allows them to be given continuously by osmotic pumps placed in the peritoneal cavity. In an attempt to increase the therapeutic index of the immunotoxins, we have now delivered them continuously for 6 days through mini-osmotic pumps placed in the peritoneal cavity of tumor-bearing nude mice. Using this mode of administration, we were able to maintain a constant level of immunotoxin in the serum which was non-toxic to the mice, but caused complete regressions of large 150-to 200-mm³ tumors which lasted for over a month at 1/11 of the LD₅₀ with B1(dsFv)-PE38 and 1/6 of the LD₅₀ with B3(dsFv)-PE38. Complete regression of tumors of similar size could also be achieved by i.v. bolus injections of these immunotoxins at 1/7 of the LD, o with B1 (dsFv)-PE38) and 1/3 of the LD₅₀ with B3(dsFv)-PE38. These results suggest that in patients it may be advantageous to administer dsFv-immunotoxins by continuous infusion, since a larger therapeutic index is achieved. © 1995 Wiley-Liss Inc.     

Synergistic antitumor activity of taxol and immunotoxin SS1P in tumor-bearing mice.

PURPOSE: To investigate the combined antitumor activity in mice of immunotoxin SS1P and Taxol. Methods: Immunodeficient mice were implanted with A431/K5 tumors expressing mesothelin. Established tumors were treated i.v. with immunotoxin SS1P alone, i.p. with Taxol alone, or with the two agents together. SS1P was radiolabeled with (111)In and used to study the effect of Taxol on its uptake by A431/K5 tumors. RESULTS: Using doses at which either agent alone caused stabilization of tumor growth, the combination was synergistic causing long-lasting complete remissions in many animals. In contrast, synergy was not observed when the same cells were treated with these agents in vitro. Tumor uptake of (111)In-SS1P was not affected by treatment with Taxol. CONCLUSION: The combination of Taxol and SS1P exerts a synergistic antitumor effect in animals but not in cell culture. This effect is not secondary to increased tumor uptake of the immunotoxin. Synergy could be due to improved immunotoxin distribution within the tumor or could involve factors released by other cell types in the tumors.     

Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2.

We have generated a stable bivalent Fv molecule [(dsFv)2] of the anti-erbB2 monoclonal antibody e23 in which the V(H) and V(L) domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a 15-amino acid linker (T. K. Bera et al., J. Mol. Biol., 281: 475-483, 1998). The e23 (dsFv)2 molecule is linked to a truncated form of Pseudomonas exotoxin (PE38) to generate a bivalent disulfide-stabilized immunotoxin e23 (dsFv)2-PE38. Compared to the monovalent immunotoxin, the (dsFv)2 immunotoxin showed greatly increased cytotoxicity to four cancer cell lines expressing low levels of erbB2 but not to four other cell lines with high erbB2 expression. e23 (dsFv)2-PE38 was administered i.v. to mice, and its half-life was determined. The t(1/2)alpha and t(1/2)beta were 20 and 325 min, respectively, whereas the corresponding values for the monovalent dsFv immunotoxin were shorter, 6 and 52 min. The antitumor activities of the monovalent and bivalent immunotoxin were compared using mice bearing A431 tumors. Despite the fact that e23 (dsFv)2-PE38 was 13-fold more active than e23 dsFv-PE38 on A431 cells in cell culture, its antitumor activity in mice was <2-fold that of the monovalent immunotoxin. These data show that a large increase in avidity does not always lead to an increase in cytotoxic activity. Furthermore, in one of the cases in which cytotoxic activity in vitro was greatly enhanced, there was only a small increase in antitumor activity.     

Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.     

Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-TAC Fv fragment and truncated pseudomonas exotoxin

Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the V_H-V_L heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of V_H and V_L. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the α subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37° C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disu Hide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications. © 1994 Wiley-Liss, Inc.     

Mesothelin targeted cancer immunotherapy

Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is, however, highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian and lung adenocarcinoma. The normal biologic function of mesothelin is unknown but recent studies have shown that it binds to CA-125 and may play a role in the peritoneal spread of ovarian cancer. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. Three mesothelin targeted agents are in various stages of clinical evaluation in patients. These include SS1P (CAT-5001) a recombinant immunotoxin targeting mesothelin, MORAb-009 a chimeric anti-mesothelin monoclonal antibody and CRS-207 a live-attenuated Listeria monocytogenes vector encoding human mesothelin. These ongoing clinical trials will help define the utility of mesothelin as a target for cancer therapy.     

In vitro and in vivo cytotoxic activities of recombinant immunotoxin 8H9(Fv)-PE38 against breast cancer, osteosarcoma, and neuroblastoma

The 8H9 monoclonal antibody (MAb) is highly reactive with a cell surface glycoprotein expressed on human breast cancers, childhood sarcomas, and neuroblastomas but is not reactive with the cell surface of normal human tissues. This specific reactivity suggests that MAb 8H9 may be useful for targeted cancer therapy. To explore this possibility, we generated two recombinant immunotoxins (ITs) using the single-chain Fv (scFv) of MAb 8H9. Initially the 8H9(scFv) cDNA was fused to a DNA encoding a 38-kDa truncated form of Pseudomonas exotoxin (PE38) to generate the IT 8H9(scFv)-PE38. The fusion gene was expressed in Escherichia coli, and the IT was purified to near homogeneity from inclusion bodies. The purified IT showed specific cytotoxicity on nine different cancer cell lines derived from breast cancer, osteosarcoma, and neuroblastomas, known to react with MAb 8H9. The cytotoxic activity was inhibited by MAb 8H9, showing the cytotoxic activity is specific. The antitumor activity of 8H9(scFv)-PE38 was evaluated in severe combined immunodeficient mice bearing MCF-7 breast cancers or OHS-M1 osteosarcomas. The IT showed a specific dose-dependent antitumor activity at 0.075 and 0.15 mg/kg. Next, a more stable disulfide-linked IT, 8H9(dsFv)-PE38, was constructed. It was produced in high yield (16%) and showed cytotoxic and antitumor activities similar to those of 8H9(scFv)-PE38. 8H9(dsFv)-PE38 was given to two cynomolgus monkeys at doses of 0.1 and 0.2 mg/kg i.v. QOD x 3 and was well tolerated. This shows that a dose that causes significant tumor regressions in mice is well tolerated by monkeys. These results make 8H9(dsFv)-PE38 a candidate for further development as a therapeutic agent for breast cancers, osteosarcomas, and neuroblastomas.     

Recombinant single-chain and disulfide-stabilized Fv-immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice

Monoclonal antibody (MAb) 55.1 specifically recognizes an antigen on the surface of human colon adenocarcinoma cells. We constructed recombinant immunotoxins composed of the heavy- and light-chain variable regions of MAb 55.1 fused to a recombinant form of Pseudomonas exotoxin (PE). The heavy- and light-chain variable regions are stabilized by 2 means. One is by a flexible peptide linker to form a single-chain antigen binding protein (scFv) and the second by an interchain disulfide bond engineered between structurally conserved framework regions. These are termed disulfide stabilized Fvs (dsFv). The 2 Fv forms are fused to truncated forms of PE lacking the cell binding domain. The recombinant scFv- and dsFv-immunotoxins were expressed in E. coli and purified to near homogeneity. The scFv- and dsFv-immunotoxins were shown to be specifically cytotoxic to human colon adenocarcinoma cell lines. The scFv-immunotoxin containing PE38KDEL was more active than the immunotoxin containing PE38 with the native carboxyl terminus (REDLK). However, the PE38KDEL immunotoxin is about 2-fold more toxic in mice, and therefore it does not appreciably increase the therapeutic window in mice. Intravenous administration of the scFv- and dsFv- recombinant immunotoxins caused complete regression of a human colon carcinoma (Colo205) growing subcutaneously in immunodeficient mice. The dsFv-immunotoxin has better antitumor activity compared with its scFv-immunotoxin counterpart. © 1996 Wiley-Liss, Inc.     

Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers.

PURPOSE: To determine the toxicities, maximum tolerated dose (MTD) and pharmacokinetics of the recombinant immunotoxin SS1P (anti-mesothelin dsFv-PE38) in patients with mesothelin-expressing cancers. EXPERIMENTAL DESIGN: SS1P given as a 30-min i.v. infusion every other day (QOD) for six or three doses was administered to 34 patients with advanced mesothelioma (n = 20), ovarian (n = 12), and pancreatic (n = 2) cancer. RESULTS: The initial cohort of 17 patients received SS1P QOD x 6 doses and the MTD was 18 microg/kg/dose. Dose-limiting toxicities (DLT) included grade 3 uticaria (one patient) and grade 3 vascular leak syndrome (two patients). To allow further SS1P dose escalation, 17 patients were treated on the QOD x 3 schedule and the MTD was 45 microg/kg/dose. The DLT was grade 3 pleuritis and was seen in two of two patients treated at a dose of 60 microg/kg and in one of nine patients treated at a dose of 45 microg/kg. At the MTD of 45 microg/kg, the mean C(max) of SS1P was 483 ng/mL and half-life was 466 min. Of the 33 evaluable patients treated, 4 had minor responses, 19 had stable disease (including 2 with resolution of ascites), and 10 had progressive disease. CONCLUSIONS: SS1P is well tolerated with pleuritis as the DLT at the highest dose level. Evidence of clinical activity was noted in a group of heavily pretreated patients. Phase II clinical trials of SS1P are being planned for malignant mesothelioma and other mesothelin-expressing malignancies.     

Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer.

PURPOSE: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. EXPERIMENTAL DESIGN: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. RESULTS: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1 and was undetectable by postoperative day 7. CONCLUSIONS: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.     

Cytotoxic activity of immunotoxin SS1P is modulated by TACE-dependent mesothelin shedding

Mesothelin is a cell-surface tumor-associated antigen expressed in several human cancers. The limited expression of mesothelin on normal tissues and its high expression in many cancers make it an attractive candidate for targeted therapies using monoclonal antibodies, immunoconjugates, and immunotoxins. Mesothelin is actively shed from the cell surface and is present in the serum of patients with malignant mesothelioma, which could negatively affect the response to these therapies. We have found that mesothelin sheddase activity is mediated by a TNF-α converting enzyme (TACE), a member of the matrix metalloproteinase/a disintegrin and metalloprotease family. We showed that EGF and TIMP-3 act through TACE as endogenous regulators of mesothelin shedding. We also found that reducing shedding significantly improved the in vitro cytotoxicity of immunotoxin SS1P, which targets mesothelin and is currently in clinical trials for the treatment of patients with mesothelioma and lung cancer. Our findings provide a mechanistic understanding of mesothelin shedding and could help improve mesothelin-based targeted therapies.     

Investigation of a plasmid containing a novel immunotoxin VEGF165‐PE38 gene for antiangiogenic therapy in a malignant glioma model

Inhibition of tumor neovascularization has profound effects on the growth of solid tumors. Our previous studies have shown the effect of VEGF165‐PE38 recombinant immunotoxin on proliferation and apoptosis in human umbilical vein endothelial cells in vitro. In this study, we explored the direct inhibition of angiogenesis in chick chorioallantoic membrane and antiangiogenic therapy in a malignant glioma model. HEK293 cells were transfected with the pVEGF165PE38‐IRES2‐EGFP plasmid. ELISA was used to confirm the expression of VEGF165‐PE38 in the transfected cells. These cells released 1396 ± 131.9 pg VEGF165‐PE38/1×10⁴ cells/48 h into the culture medium and the supernatant was capable of inhibiting the growth of capillary‐like structures in chick chorioallantoic membrane assay. In a murine malignant glioma model, plasmid was directly administered via multiple local intratumoral delivery. After day 16 the tumor volume in mice treated with pVEGF165PE38‐IRES2‐EGFP was significantly lower than that in mice in the control groups. Immunohistochemistry studies showed that the treated group had decreased expression of CD31. Quantitative analysis of microvessel density in the treated group was 1.99 ± 0.69/0.74 mm², and was significantly lower than that in the control groups (9.33 ± 1.99/0.74 mm², 8.09 ± 1.39/0.74 mm² and 8.49 ± 1.69/0.74 mm²). Immunohistochemistry analysis indicated that immunotoxin VEGF165‐PE38 was distributed in the treated group in malignant glioma tissue. Our findings provide evidence that the in vivo production of VEGF165‐PE38 through gene therapy using a eukaryotic expression plasmid had potential antiangiogenic activity in malignant glioma in vivo.     

Antitumor activity of an immunotoxin in a nude mouse model of human ovarian cancer

An immunotoxin composed of an antibody to the human transferrin receptor (454A12) and ricin A chain (RTA) was shown to inhibit the growth of NIH:OVCAR-3 tumors in a nude mouse model of human ovarian cancer. Inhibition of tumor growth by 454A12-RTA was related to the dose administered. The antitumor activity of the immunotoxin was blocked by coinjection of excess antibody with immunotoxin. An immunotoxin made using 454A12 and recombinant ricin A chain (rRTA) had an activity similar to that made with native RTA. The administration of 10 micrograms or greater of the immunotoxin 454A12-RTA/rRTA had significant antitumor activity. The injection of 30 micrograms of an irrelevant immunotoxin, MOPC21-RTA, or 30 to 500 micrograms of the 454A12 antibody had no antitumor activity.     

Apoptosis induced by immunotoxins used in the treatment of hematologic malignancies

The recombinant immunotoxins anti-Tac(Fv)-PE38 (LMB-2), targeting the interleukin-2 receptor alpha subunit (IL-2Ralpha, Tac or CD25), and RFB4(dsFv)-PE38 (BL22), targeting CD22, are being evaluated in clinical trials as treatment for hematologic malignancies. The toxin moiety Pseudomonas exotoxin A (PE) of these recombinant molecules leads to the arrest of protein synthesis due to inactivation of elongation factor 2. Here, we provide evidence that cell lines derived from patients with hematologic malignancies react to immunotoxins not only with inhibition of protein synthesis but also with characteristic hallmarks of apoptosis such as caspase activation, cleavage of the "death substrate poly(ADP)-ribose polymerase and DNA laddering. Anti-Tac(Fv)-PE38 leads to a 10-fold increase in the cleavage of the fluorescent substrate DEVD-AFC, suggesting that a caspase-3-like enzyme is involved. This was verified by cleavage of caspase-3 (CPP32). MT1 cells exhibited DNA laddering after treatment with immunotoxin, which was reversed by pre-treatment with the protease inhibitor zVAD-fmk. This caspase inhibitor led to an at least 5-fold improvement in cell viability without altering inhibition of protein synthesis. Interestingly, HUT-102 cells did not undergo programmed cell death after exposure to immunotoxins that kill these cells. We conclude that immunotoxins may be valuable in the treatment of cancers that are resistant toward apoptosis because their targeted killing is often facilitated by, but not completely dependent on, programmed cell death. Int. J. Cancer 87:86-94, 2000. Published 2000 Wiley-Liss, Inc.     

Identification of novel human CTL epitopes and their agonist epitopes of mesothelin.

PURPOSE: Mesothelin is overexpressed in many pancreatic and ovarian cancers, mesotheliomas, and other tumor types. Clinical trials are ongoing using immunotoxins to target mesothelin, and patients immunized with allogeneic pancreatic tumor cell lines have shown immune responses to previously defined mesothelin epitopes. The purpose of this study was to define novel mesothelin CTL epitopes and, more importantly, agonist epitopes that would more efficiently activate human T cells to more efficiently lyse human tumors. EXPERIMENTAL DESIGN AND RESULTS: Two novel mesothelin HLA-A2 epitopes were defined. T-cell lines generated from one of these epitopes were shown to lyse pancreatic and ovarian tumor cells. Several agonist epitopes were defined and were shown to (a) have higher affinity and avidity for HLA-A2, (b) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-gamma and the chemokine lymphotactin, and (c) lyse several mesothelin-expressing tumor types in a MHC-restricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells. CONCLUSIONS: The identification of novel CTL agonist epitopes supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic and ovarian cancers, as well as mesotheliomas.